Fasudil in combination therapies for the treatment of pulmonary arterial hypertension

ABSTRACT

Preferred embodiments of the present invention are related to novel therapeutic drug combinations and methods for treating and/or preventing pulmonary arterial hypertension and/or stable angina. More particularly, aspects of the present invention are related to therapeutic combinations comprising a Rho-kinase inhibitor, such as fasudil, and one or more additional compounds selected from the group consisting of prostacyclins, such as iloprost, endothelin receptor antagonists, PDE inhibitors, calcium channel blockers, 5-HT 2A  antagonists, such as sarpogrelate, selective serotonin reuptake inhibitors, such as fluoxetine, statins, and vascular remodeling modulators, such as Gleevec.

RELATED APPLICATIONS

The present application is a continuation of pending U.S. patentapplication Ser. No. 11/588,185, filed on Oct. 25, 2006, which claimsthe benefit of U.S. Provisional Patent Application No. 60/730,273, filedon Oct. 26, 2005. The disclosures of application Ser. Nos. 11/588,185and 60/730,273 are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

Embodiments of this invention are related to therapeutic formulationsand methods of using Rho-kinase inhibitors, such as fasudil, incombination with a prostacyclin or prostacyclin agonist, such asiloprost for treating and/or preventing pulmonary arterial hypertension(“PAH”). In other preferred embodiments, the Rho-kinase inhibitor may beused in therapeutic combinations with other agents, such as for example,endothelin receptor antagonists, PDE inhibitors, calcium channelblockers, 5-HT_(2A) antagonists (such as sarpogrelate), selectiveserotonin reuptake inhibitors (such as fluoxetine), statins, andvascular remodeling modulators (such as Gleevec) for the treatmentand/or prevention of PAH and/or stable angina.

BACKGROUND OF THE INVENTION

Pulmonary arterial hypertension is a debilitating disease characterizedby an increase in pulmonary vascular resistance leading to rightventricular failure and death. PAH with no apparent cause is termedprimary pulmonary hypertension (“PPH”). Recently, variouspathophysiological changes associated with this disorder, includingvasoconstriction, vascular remodeling (i.e. proliferation of both mediaand intima of the pulmonary resistance vessels), and in situ thrombosishave been characterized (e.g., D'Alonzo, G. E. et al. 1991 Ann InternMed 115:343-349; Palevsky, H. I. et al. 1989 Circulation 80:1207-1221;Rubin, L. J. 1997 N Engl J Med 336:111-117; Wagenvoort, C. A. &Wagenvoort, N. 1970 Circulation 42:1163-1184; Wood, P. 1958 Br Heart J20:557-570). Impairment of vascular and endothelial homeostasis isevidenced from a reduced synthesis of prostacyclin (PGI₂), increasedthromboxane production, decreased formation of nitric oxide andincreased synthesis of endothelin-1 (Giaid, A. & Saleh, D. 1995 N Engl JMed 333:214-221; Xue, C & Johns, R. A. 1995 N Engl J Med 333:1642-1644).The intracellular free calcium concentration of vascular smooth musclecells of pulmonary arteries in PPH has been reported to be elevated.

The pathogenesis of pulmonary hypertension (“PH”) is a complex andmultifactorial process. Pathologic changes of pulmonary arteries, whichinvolve endothelial dysfunction, endothelial and smooth muscle cellproliferation, and increased vasoconstriction, decrease the lumen areaof the pulmonary microvasculature, causing fixed elevation of pulmonaryvascular resistance. Although these pathological features are common toall forms of human PH, the mechanisms responsible for this abnormalvascular proliferation are unknown. However, impairment of endothelialfunctions leading to an imbalance of vasodilator and vasoconstrictorinfluences is likely to play a central role in the initiation andprogression of PH. Drugs that improve the endothelial function orrestore the altered balance of endothelium-derived vasoactive mediatorssuch as endothelin-1 receptor antagonists and prostacyclin analogues areused to treat this disease with moderate success. The NO/cGMP axis isalso considered as a major target for the treatment of PH. Recently, thetype 5 phosphodiesterase inhibitor sildenafil has been identified as apromising therapeutic agent for PH. The type 5 phosphodiesterase is themajor cGMP-degrading phosphodiesterase in the pulmonary vasculature andis upregulated in PH. Sildenafil reduces right ventricular hypertrophyin chronic hypoxic mice, pulmonary arterial pressure in chronic hypoxicrats, and improves survival rate in rats with PH induced bymonocrotaline injection. Short-term studies in patients with PH suggestthat sildenafil is an effective pulmonary vasodilator.

On the other hand, recent pharmacological studies have suggested a rolefor the serine/threonine kinase Rho kinase in the development of PH. Invivo, intravenous or oral treatment with Rho kinase inhibitor (Y-27632or fasudil) nearly normalizes the high pulmonary arterial pressure inchronically hypoxic rats, attenuates the development of chronichypoxia-induced PH in mice, and reduces pulmonary arterial lesions inthe model of monocrotaline-induced PH in rats (Abe K. et al. 2004 Circ.Res. 94:385-393; Fagan K. A. et al. 2004 Am. J. Physiol. Lung Cell. Mol.Physiol. 287:L656-L664; Nagaoka et al., 2004 Am. J. Physiol. Lung CellMol. Physiol. 287:L665-L672). In addition, inhaled Y-27632 or fasudilcauses sustained and selective pulmonary vasodilation inmonocrotaline-induced PH and in spontaneous PH in fawn-hooded rats, aswell as in chronically hypoxic rats (Nagaoka et al. 2005 Am. J. Respir.Crit. Care Med. 171:494-499). Rho kinase is one of the main downstreameffectors of the small G protein RhoA, which functions as a tightlyregulated molecular switch that governs a wide range of cellularfunctions (Van Aelst & D'Souza-Schorey, 1997 Genes Dev. 11:2295-2322).In particular, Rho kinase phosphorylates the myosin phosphatase targetsubunit 1 (MYPT1) of smooth muscle myosin phosphatase at Thr-696,leading to the inhibition of its activity. This inhibition of smoothmuscle myosin phosphatase activity is a primary mechanism of the Ca²⁺sensitization of smooth muscle contraction. A large body of evidence hasnow been obtained regarding the important functions of RhoA in thevasculature, and RhoA has been shown to play a major role in theregulation of vascular cell processes such as actin cytoskeletonorganization, contraction, gene expression, and differentiation. Invascular smooth muscle cells, RhoA has been shown to be regulated by theNO/cGMP pathways. RhoA is phosphorylated by cGMP-dependent proteinkinase (PKG) (Sauzeau et al., 2000 J. Biol. Chem. 275:21722-21729). Thisphosphorylation prevents the translocation of active GTP-bound RhoA tothe membrane, which is an obligatory step for the activation of itsdownstream effectors. Activation of the NO/cGMP pathway thus leads tothe inhibition of RhoA-dependent functions, including actin cytoskeletonorganization, Ca²⁺ sensitization of the contraction, and genetranscription (Sauzeau et al., 2000 J. Biol. Chem. 275:21722-21729; Gudiet al., 2002 Biol. Chem. 277:37382-37393). The beneficial effect of Rhokinase inhibitor on PH could be ascribed to multiple mechanisms,including its inhibitory effect on pulmonary vasoconstriction (Robertsonet al., 2000 Br. J. Pharmacol. 131:5-9; Wang et al., 2001 Am. J. Respir.Cell Mol. Biol. 25:628-635; Nagaoka et al., 2004 Am. J. Physiol. LungCell Mol. Physiol. 287:1,665-L672), the prevention of mechanicalstress-induced expression of growth factors (Wilson et al., 1993 J. CellBiol. 123:741-747), or the inhibition of Rho kinase-mediated mitogeniceffects of serotonin (Liu et al., 2004 Circ. Res. 95: 579-586) and Rhokinase-mediated inhibition of nitric oxide synthase (Takemoto et al.,2002 Circulation 106: 57-62).

Current therapies for pulmonary hypertension are unsatisfactory. Thesetypically involve calcium channel antagonists, prostacyclins, endothelinreceptor antagonists and long-term anticoagulant therapy. However, eachtreatment has limitations and side effects.

Consequently there is a long felt need for a new and combined medicamentfor the treatment of PAH, preferably employing lower doses of the activeagents, which exhibits fewer or no adverse effects (i.e., less toxicity)and a favorable profile in terms of effectiveness in patients indifferent stages of PAH.

SUMMARY OF THE INVENTION

A therapeutic combination is disclosed herein for the treatment and/orprevention of PAH and/or angina. The disclosed combinations comprise aneffective amount of a Rho-kinase inhibitor and at least one additionalcompound selected from the group consisting of prostacyclins, endothelinreceptor antagonists, PDE inhibitors, calcium channel blockers,5-HT_(2A) antagonists, selective serotonin reuptake inhibitors, statins,and vascular remodeling modulators.

In preferred embodiments, the Rho-kinase inhibitor is selected from thegroup consisting of fasudil, H-1152P, and Y-27632.

In preferred embodiments, the prostacyclin is selected from the groupconsisting of iloprost, treprostinol, and beraprost.

In preferred embodiments, the endothelin receptor antagonist is selectedfrom the group consisting of bosentan, sitaxentan, and ambrisentan.

In preferred embodiments, the PDE inhibitor is selected from the groupconsisting of enoximone, milrinone, aminone, sildenafil, tadalafil andvardenafil.

In preferred embodiments, the calcium channel blocker is selected fromthe group consisting of amlodipine, diltiazem, isradipine, nicardipine,nifedipine, nimodipine, nisoldipine, nitrendipine, and verapamil.

In preferred embodiments, the 5-HT_(2A) antagonist is sarpogrelate.

In preferred embodiments, the selective serotonin reuptake inhibitor isselected from the group consisting of fluoxetine, sertrlaine, paroxetineand venlafaxine.

In preferred embodiments, the statin is selected from the groupconsisting of fluvastatin, pitavastatin, pravastatin and atorvastatin.

In preferred embodiments, the vascular remodeling modulator is Gleevec.

In preferred embodiments, the Rho-kinase inhibitor and at least oneadditional compound may be formulated together or independently.

Most preferably, the Rho-kinase inhibitor is fasudil.

The fasudil may be formulated for administration via inhalation. Forexample, the fasudil may be formulated in a dry powder or anaerosolizable solution.

A therapeutic combination is disclosed in accordance with one preferredembodiment. The combination comprises fasudil and iloprost or saltsthereof, wherein the fasudil and iloprost, or salts thereof are providedin amounts which together are sufficient to treat and/or prevent atleast one symptom associated with PAH. At least one of fasudil andiloprost are preferably formulated for administration by inhalation.Alternatively, both fasudil and iloprost may be formulated foradministration by inhalation. In preferred combinations, at least oneadditional compound is included, selected from the group consisting ofendothelin receptor antagonists, PDE inhibitors, calcium channelblockers, selective serotonin reuptake inhibitors, statins, and vascularremodeling modulators.

Another therapeutic combination is disclosed, comprising fasudil andsarpogrelate or salts thereof, wherein the fasudil and sarpogrelate orsalts thereof are provided in amounts which together are sufficient totreat and/or prevent at, least one symptom associated with PAH and/orstable angina. Preferably, at least one of fasudil and sarpogrelate areformulated for administration by inhalation. Alternatively, both fasudiland sarpogrelate are formulated for administration by inhalation. Inpreferred combinations, at least one additional compound is included,selected from the group consisting of endothelin receptor antagonists,PDE inhibitors, calcium channel blockers, selective serotonin reuptakeinhibitors, statins, and vascular remodeling modulators.

A method of treating and/or preventing PAH and/or stable angina is alsodisclosed. The method comprises administering effective amounts of anyof the therapeutic combinations of claim 1, 15 or 18.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A, B and C) shows dose response curves of effects of fasudil(A), iloprost (B) and fasudil after iloprost, 10⁻⁶ M (C) on relaxationof vasoconstricted small pulmonary arteries rings.

FIG. 2 (A, B and C) shows dose response curves of effects of fasudil(A), bosentan (B) and fasudil after bosentan, 5×10⁻⁵ M (C) on relaxationof vasoconstricted small pulmonary arteries rings.

FIG. 3 (A, B and C) shows dose response curves of effects of fasudil(A), Viagra™ (B) and fasudil after Viagra™, 10⁻⁵ M (C) on relaxation ofvasoconstricted small pulmonary arteries rings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In an embodiment of the present invention, a combination therapy isdisclosed for treating and/or preventing PAH. In one preferredembodiment, the combination therapy comprises a therapeuticallyeffective amount of a Rho-kinase inhibitor, and most preferably fasudil,in combination with a therapeutically effective amount of at least oneadditional active agent. In one embodiment, the Rho-kinase inhibitor iscombined with a prostacyclin, most preferably, iloprost. In anotherembodiment, the Rho-kinase inhibitor and prostacyclin are furthercombined with one or more additional active agents. The active agentsmay be formulated and administered together or may be formulated andadministered independently. For example, the at least one additionalactive agent may be formulated together with the Rho-kinase inhibitor ina single tablet or capsule, a single dry powder formulated forinhalation, or a solution formulated for aerosolization and inhalation.Any of the additional agents may be formulated and administeredseparately from the Rho-kinase inhibitor. In one preferred embodiment,the Rho-kinase inhibitor is aerosolized. In one preferred embodiment,the Rho-kinase inhibitor is administered by any route, and aprostacyclin is administered by inhalation, e.g., dry powder oraerosolized formulation. In one preferred embodiment, therapeuticallyeffective amounts of both fasudil and iloprost are combined andadministered together via inhalation in dry powder or aerosol form.

In one embodiment, the Rho-kinase inhibitor may be combined with one ormore additional active agents selected from the group consisting ofprostacyclins, endothelin receptor antagonists, PDE inhibitors, calciumchannel blockers, 5-HT_(2A) antagonists, selective serotonin reuptakeinhibitors, statins, and vascular remodeling modulators. Preferably, theone or more additional active agents used in the therapeutic combinationmodulates pulmonary arterial pressure through a mechanism which isdistinct from that of fasudil. Preferably, the prostacyclin is selectedfrom the group consisting of treprostinol (Remodulin®, UnitedTherapeutics), beraprost, and iloprost (Ventavis®). Preferably, theendothelin receptor antagonist is selected from the group consisting ofbosentan (Tracleer™, Actelion), ambrisentan (Myogen) and sitaxentan(Encysive Pharmaceuticals). Preferably, the PDE inhibitor is selectedfrom the group consisting of enoximone, milrinone (Primacor®), aminone(Inocor®), sildenafil (Viagra®), tadalafil (Cialis®) and vardenafil(LEVITRA®). Preferably, the calcium channel blocker is selected from thegroup consisting of amlodipine, diltiazem, isradipine, nicardipine,nifedipine, nimodipine, nisoldipine, nitrendipine, and verapamil.Preferably, the 5-HT_(2A) antagonist is sarpogrelate. Preferably, theselective serotonin reuptake inhibitor (SSRI) is selected from the groupconsisting of fluoxetine, sertrlaine, paroxetine and venlafaxine.Preferably, the statin is selected from the group consisting offluvastatin, pitavastatin, pravastatin and atorvastatin. Preferably, thevascular remodeling modulator is Gleevec.

In other preferred embodiments, therapeutic combinations are disclosedfor treatment and/or prevention of stable angina. These combinationspreferably include a Rho-kinase inhibitor, preferably fasudil, andsarpogrelate.

Rho-Kinase Inhibitors

Rho-kinase is involved in such processes as tumor invasion, celladhesion, smooth muscle contraction, and formation of focal adhesionfibers, as revealed using inhibitor Y-27632. Another Rho-kinaseinhibitor, 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077 orFasudil), is currently used in the treatment of cerebral vasospasm; therelated nanomolar inhibitor H-1152P improves on its selectivity andpotency. It was recently found that RhoA/Rho kinase signaling isinvolved in both vasoconstriction and vascular remodeling in the mousemodel of hypoxic pulmonary hypertension (Fagan L. A. et al. 2004 Am. J.Physiol. Lung Cell Mol. Physiol. 287:L656-L664), and that Rhokinase-mediated vasoconstriction substantially contributes to thesustained elevation of pulmonary vascular resistance in rat model ofhypoxic pulmonary hypertension (Nagaoka et al., 2004 Am. J. Physiol.Lung Cell Mol. Physiol. 287:L665-L672). In the latter study, the acuteeffect of intravenous Y-27632, a selective Rho-kinase inhibitor, inchronically hypoxic rats was striking (i.e., it nearly normalized theelevated pulmonary artery pressure). However, intravenous Y-27632 had nopulmonary vascular selectivity and also caused systemic vasodilation.Uehata et al. (1997, Nature 389:990-994) have reported that oraladministration of Y-27632 has potent hypotensive effects in rat modelsof systemic hypertension, but has a smaller transient effect innormotensive rats. Nagaoka et al. (2005 Am. J. Respir. Crit. Care Med.171:494-499) reported that 5 minutes of inhaled Y-27632 decreased meanpulmonary arterial pressure without reducing mean systemic arterialpressure. The hypotensive effect of inhaled Y-27632 on hypoxic pulmonaryhypertension was greater that that of inhaled nitric oxide, and theeffect lasted for at least 5 hrs. Fasudil (Asahi Kasei Pharma Co.,Tokyo, Japan) is also a Rho-kinase inhibitor that is metabolized in theliver to a more specific Rho-kinase inhibitor, hydroxyfasudil, afteroral administration in vivo (Shimokawa H. et al. 1999 Cardiovasc. Res.43:1029-1039). Inhaled fasudil caused selective mean pulmonary arterialpressure reductions in monocrotaline-induced pulmonary hypertension andin spontaneous PH in rats, as well as in chronically hypoxic rats. Inpatients with severe pulmonary hypertension, fasudil, administeredintravenously, significantly reduced pulmonary vascular resistancewithout side effects (Fukumoto Y. et al. 2005 Heart 91:391-392).

Improved HA-1077 analog,(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine(H-1152P), which is a more selective inhibitor of Rho-kinase, with aK(i) value of 1.6 nM for Rho-kinase was synthesized by Sasaki et al.(2002 Pharmacol Ther. 93:225-32). Additional specific Rho-kinaseinhibitors have been synthesized by Tamura et al. (2005 Biochim.Biophys. Acta).

The role of Rho-kinase in cardiovascular medicine and in the treatmentof stable angina is described by Shimokawa and Takeshita 2005(Arterioscler Thromb Vasc Biol 25:X-X) and Hirooka and Shimokawa 2005(Am J Cardiovasc Drugs 5(1): 31-39); these references are incorporatedherein in their entirety by reference thereto.

HMG-CoA reductase inhibitors, known as statins, have been shown tosuppress Rho-kinase activity in some systems. See e.g., Tramontano etal. 2004 (Biochem Biophys Res Comm 320: 34-38) and Ohnaka et al. 2001(Biochem Biophys Res Comm 287: 337-342); incorporated herein in theirentirety by reference thereto. Accordingly, statins such as fluvastatin,pitavastatin, pravastatin, atorvastatin, etc., may be useful inpotentiating or otherwise advantageously modulating the therapeuticeffect of the Rho-kinase inhibits disclosed herein.

Epoprostenol Derivatives

A continuous infusion of prostacyclin (Flolan®, GlaxoSmithKline) was thefirst therapy shown to reduce mortality in a controlled study ofpatients with severe pulmonary hypertension. However, its use isassociated with a number of serious drawbacks (Barst R. J. et al. 1996 NEngl J Med 334:296-301; Badesch D. B. et al. 2000 Ann Intern Med132:425-434). The lack of pulmonary selectivity results in systemic sideeffects, tolerance leads to progressive increases in the dose, and theremay be recurrent infections of the intravenous catheter. As analternative, inhaled nitric oxide possesses pulmonary selectivity, butit is less potent than prostacyclin in the pulmonary vasculature.Moreover, an interruption in the inhalation of continuous nitric oxidemay cause rebound pulmonary hypertension. Designed to combine thebeneficial effects of prostacyclin with those of an inhalationalapplication, aerosolized prostacyclin was found to be a potent pulmonaryvasodilator in patients with acute respiratory failure, exertingpreferential vasodilatation in well-ventilated lung regions (Walmrath D.et al. 1993 Lancet 342:961-962; Walmrath D. et al. 1995 Am J Respir CritCare Med 151:724-730; Walmrath D. et al. 1996 Am J Respir Crit Care Med153:991-996; Zwissler B. et al. 1996 Am J Respir Crit Care Med154:1671-1677). Similar results were obtained in spontaneously breathingpatients who had lung fibrosis and severe pulmonary hypertension(Olschewski H. et al. 1999 Am J Respir Crit Care Med 160:600-607).

Three epoprostenol analogs have been studied in the treatment of PAH:treprostinol (Remodulin®, United Therapeutics), beraprost, and iloprost.Treprostinol is a stable analogue of epoprostenol, which is givencontinuously subcutaneously. Escalation of dosage has been limited bysignificant infusion site pain. Thus many patients do not receivetherapeutic doses. Beraprost is active orally and has shown a benefit ina study in PAH at 3 and 6 months but not at 9 or 12 months (Barst, R. J.2003 J. Am. Coll. Cardiol. 41:2119-25. Iloprost can be givenintravenously or by nebulizer. The advantages of the nebulizer method ofdelivery is that less of the substance reaches the systemic circulation(a “pseudoselective” pulmonary vasodilator). Iloprost is generally givensix to nine times a day, which may disrupt the patient's lifestyle;dosing frequency may be reduced by combining iloprost with an agenthaving a therapeutic effect on the pulmonary hypertension through adifferent mechanism and possibly acting synergistically.

Recently Abe K. et al. (2005 J Cardiovasc Pharmacol 45:120-124)demonstrated that prostacyclin and beraprost lack the inhibitory effecton Rho-kinase, when administered to rats, indicating that a combinationtherapy with prostacyclin and a Rho-kinase inhibitor could have furtherbeneficial effects on pulmonary hypertension.

Iloprost

Iloprost (see U.S. Pat. No. 4,692,464; incorporated herein in itsentirety by reference thereto) is a stable analogue of prostacyclin thatis associated with a longer duration of vasodilatation (Fitscha P. etal. 1987 Adv Prostaglandin Thromboxane Leukot Res 17:450-454). Whenadministered by aerosolization to patients with pulmonary hypertension,its pulmonary vasodilative potency was similar to that of prostacyclin,but its effects lasted for 30 to 90 minutes, as compared with only 15minutes for the prostacyclin (Hoeper M. M. et al. 2000 J Am Coll Cardiol35:176-182; Olschewski H. et al. 1999 Am J Respir Crit Care Med160:600-607; Olschewski H. et al. 1996 Ann Intern Med 124:820-824;Gessler T. et al. 2001 Eur Respir J 17:14-19; Wensel R. et al. 2000Circulation 101:2388-2392). Several open-label, uncontrolled studies ofpatients with severe pulmonary hypertension suggested that long-term useof aerosolized iloprost results in substantial clinical improvement(Olschewski H. et al. 1999 Am J Respir Crit Care Med 160:600-607;Olschewski H. et al. 1996 Ann Intern Med 124:820-824; Hoeper M. M. etal. 2000 N Engl J Med 342:1866-1870; Olschewski H. et al. 1998 IntensiveCare Med 24:631-634; Stricker H. et al. 1999 Schweiz Med Wochenschr129:923-927; Olschewski H. et al. 2000 Ann Intern Med 132:435-443;Beghetti M. et al. 2001 Heart 86:E10-E10). A multi-center randomizedplacebo controlled study of patients with severe PAH has demonstratedimproved exercise capacity in patients receiving iloprost versus, thosereceiving placebo (Olschewski H et al 2002 NEJM 347:322-9).

Sarpogrelate

Sarpogrelate ((φ-amino-alkoxy)phenyl-ethylethylbenzene) is a serotoninblocker, and more particularly, a selective 5-hydroxytryptamine receptorsubtype 2A (5-HT_(2A)) antagonist. It is metabolized to racemic M-1 andboth enantiomers of M-1 are also antagonists of 5-HT_(2A) receptors.Sarpogrelate inhibits responses to 5-HT mediated by 5-HT_(2A) receptorssuch as platelet aggregation, vasoconstriction and vascular smoothmuscle proliferation. Sarpogrelate is efficacious in animal models ofthrombosis, coronary artery spasm, atherosclerosis, restenosis,peripheral vascular disease, pulmonary hypertension, ischemic heartdisease, myocardial infarction, diabetes and kidney disease (Doggrell S.A. 2004 Expert Opin. Investig. Drugs 13:7865-874). In the commonly usedmodel of pulmonary hypertension, induced by monocrotaline in rat, theseverity of pulmonary hypertension, determined by the medial thicknessof the small pulmonary arteries and right ventricle/left ventricle andseptum ration, were reduced by treatment with sarpogrelate (Miyata M. etal. 2000 Lung 178:63-73). Sarpogrelate also reduced the thickening ofthe alveolar walls and interstitial inflammatory cell infiltration. Thenumber of proliferating cell nuclear antigen-positive cells was alsoreduced by sarpogrelate (Miyata M. et al. 2000 Lung 178:63-73).

The benefit of sarpogrelate in the monocrotaline model of pulmonaryhypertension has been confirmed. Sarpogrelate, immediately followingmonocrotaline injection, suppressed the severe pulmonary vascularremodeling and right side heart failure and reduced mortality (HironakaE. et al. 2003 Cardiovasc. Res. 60:692-629). However, late treatmentwith sarpogrelate failed to reverse established pulmonary hypertension.Sarpogrelate was also tested in a rat model of pulmonaryembolism/pulmonary hypertension. In that model, rats injected with IL-6develop extensive microarterial thrombosis in the lungs along withhypercoagulation and hyperfibrinolysis. The simultaneous sarpogrelatetreatment in the rat prevented the IL-6-induced increase in medialthickness of small pulmonary arteries and the right ventricularhypertrophy (Miyata M. et al. 2001 Chest 119:554-561; Said H. K. et al.2004 Cardiovasc. Drug ReV. 22:27-54). Sarpogrelate has no effect on thesystolic blood pressure or heart weight of spontaneously hypertensiverats (Setoguchi Y. et al. 2002 Pharmacol. 64:71-75).

In clinical trials, sarpogrelate significantly decreased respiratoryfailure and mean pulmonary arterial pressure in patients with Raynaud'sphenomenon and systemic sclerosis (Kato S. et al. 2000 Respirology5:27-32; Kato S. et al. 2000 J. Int. Med. Res. 28:258-268).

In addition, sarpogrelate has also been reported as an effectivetreatment for stable angina. Kinugawa et al., (Am Heart J 2002; 144:e1;incorporated herein in its entirety by reference thereto) had previouslydemonstrated that a single oral administration of sarpogrelate, a 5-HT2Areceptor antagonist, may improve exercise capacity in anginal patientswith well-developed collaterals. The researchers further investigatedthe effectiveness of 2-week treatment with sarpogrelate on anginalsymptoms and exercise capacity in anginal patients.

A treadmill exercise test was repeated after a 2-week period with orwithout sarpogrelate (100 mg 3 times a day) in 20 patients withangiographically proven stable angina. Anginal symptoms and dailyphysical activity by the specific activity scale (SAS) were alsoevaluated. Treatment with sarpogrelate significantly increased the SASscore and prolonged exercise time to the onset of 0.1-mV ST depression.When data were analyzed in a subgroup of patients (n=8) withwell-developed collaterals, the treatment with sarpogrelate decreasedthe number of anginal attacks (control vs sarpogrelate, 3.0±2.8 vs0.9±1.1/2 weeks, P<0.05), increased the SAS score (5.2±1.6 vs 6.2±1.3METS, P<0.05), and increased the time to the onset of 0.1-mV STdepression (235±84 vs 295±127 seconds, P<0.05). In addition, the doubleproduct at the onset of 0.1-mV ST depression increased by 15% (P<0.05)after sarpogrelate. In contrast, all parameters were not significantlychanged after sarpogrelate treatment in patients (n=12) withoutwell-developed collaterals. These findings indicate the therapeuticeffectiveness of sarpogrelate for anginal patients, especially forpatients with well-developed collaterals.

Endothelin Receptor Antagonists (ETRA)

There is increasing evidence that endothelin-1 has a pathogenic role inpulmonary arterial hypertension and that blockade of endothelinreceptors may be beneficial. Endothelin-1 is a potent endogenousvasoconstrictor and smooth-muscle mitogen that is overexpressed in theplasma and lung tissue of patients with pulmonary arterial hypertension.There are two classes of endothelin receptors: Endothelin A, ET-A andEndothelin B, ET-B receptors, which play significantly different rolesin regulating blood vessel diameter. The binding of endothelin to ET-Areceptors located on smooth muscle cells causes vasoconstriction,whereas the binding of endothelin to ET-B receptors located on thevascular endothelium causes vasodilatation through the production ofnitric oxide. This latter activity of the ET-B receptor is thought to becounter-regulatory and protects against excessive vasoconstriction.

Therefore, another attractive approach to treating pulmonaryhypertension has been the blockade of these endothelin receptors. Twotypes of ETRAs have been developed: dual ETRAs, which block thereceptors for both ET-A and ET-B, and selective ETRAs, which block onlythe ET-A receptor.

a) Dual Endothelin Receptor Antagonist

The first generation ETRAs are non-selective and block both the ET-A andET-B receptors. Bosentan (Tracleer™) is the first FDA approved ETRA (seeU.S. Pat. No. 5,292,740; incorporated herein in its entirety byreference thereto). Two placebo controlled trials of bosentan (anendothelin receptor A and B antagonist) have been conducted (Channick R.N. et al. 2001 Lancet 358:1119-1123; Rubin L. J. et al. 2002 N Engl JMed 346:896-903). The six minute walk test improved in the whole group,but the improvement was greater when the drug was used in higher doses.However, liver toxicity occurred with the higher dose.

b) Selective Endothelin Receptor Antagonist

Second generation ETRAs bind to the ET-A receptor in preference to theET-B receptor. Currently, there are two selective ETRAs in clinicaltrials: sitaxsentan and ambrisentan (BSF 208075). A pure endothelin Aantagonist, sitaxsentan has been used in an open pilot study. Thisshowed an improvement in the six minute walk test and a decrease inpulmonary vascular resistance of 30% (Bust R. J. et al. 2000 Circulation102:II-427).

A more potent endothelin compound, TBC3711 (Encysive Pharmaceuticals),entered Phase I testing in December 2001. This drug holds potential fortreating chronic heart failure and essential hypertension.

There are small clinical trials of using bosentan in patients that arealready on other medications for the treatment of pulmonary hypertension(Hoeper M. M. et al. 2003 in: “Pulmonary Hypertension: Clinical”, Abstr.A275, May 18, 2003; Pulmonary Hypertension Roundtable 2002,Phassociation.org/medical/advances in PH/spring 2002). In a preferredembodiment of the present invention, the combination therapy comprisesfasudil, iloprost, and bosentan acting in combination through distinctmechanisms of action, preferably synergistically, to treat pulmonaryhypertension. In yet another preferred embodiment, fasudil with iloprostare combined with sitaxentan. In yet another embodiment, fasudil withiloprost are combined with ambrisentan. In yet another embodimentfasudil and/or iloprost are aerosolized and administered in combinationwith bosentan, or sitaxentan, or ambrisentan. In another embodiment,fasudil and iloprost are combined with TBC3711 in combination therapy ofpulmonary hypertension.

Nitric Oxide Production

Endothelial production of nitric oxide is diminished with pulmonaryhypertension, prompting attempts to reverse this defect either by givingcontinuous inhaled nitric oxide, which is effective but difficult toadminister, or by increasing the substrate for nitric oxide L-arginine(Nagaya N. et al. 2001 Am J Respir Crit Care Med 163:887-891). A trialof supplementation with L-arginine is currently under way.

PDE Inhibitors

In addition to increasing the supply of nitric oxide, attempts todirectly increase cyclic nucleotide second messenger levels in thesmooth muscle cells have been made. Sildenafil used for erectiledysfunction blocks the enzyme phosphodiesterase type 5 present in thecorpus cavernosum of the penis and also the lungs. This raises thepossibility that a phosphodiesterase inhibitor, preferably a PDE type 5inhibitor such as sildenafil, could be a relatively selective pulmonaryvasodilator. There is empirical evidence supporting the inventor'sselection of PDE inhibitors as a target compound in a combinationtherapy (see e.g., Michelakis E. et al. 2002 Circulation 105:2398-2403;Ghofrani H. et al. 2002 Lancet 360:895-900; the disclosures of which areincorporated herein in their entirety by reference).

Although aerosolized prostacyclin (PGI₂) has been suggested forselective pulmonary vasodilation as discussed above, its effect rapidlylevels off after termination of nebulization. Stabilization of thesecond-messenger cAMP by phosphodiesterase (PDE) inhibition has beensuggested as a strategy for amplification of the vasodilative responseto nebulized PGI₂ Lung PDE3/4 inhibition, achieved by intravascular ortransbronchial administration of subthreshold doses of specific PDEinhibitors, synergistically amplified the pulmonary vasodilatoryresponse to inhaled PGI₂, concomitant with an improvement inventilation-perfusion matching and a reduction in lung edema formation.The combination of nebulized PGI₂ and fasudil with PDE3/4 inhibition maythus offer a new concept for selective pulmonary vasodilation, withmaintenance of gas exchange in respiratory failure and pulmonaryhypertension (Schermuly R. T. et al. 2000 J Pharmacol Exp Ther292:512-20). There are some reports of small clinical studies showingthat such combination therapy may be efficacious in the treatment ofpulmonary hypertension (Ghofrani et al. 2002 Crit Care Med 30:2489-92;Ghofrani et al. 2003 J Am Coll Cardiol 42:158-164; Ghofrani et al. 2002Ann Intern Med 136:515-22).

Isozymes of cyclic-3′,5′-nucleotide phosphodiesterase (PDE) are acritically important component of the cyclic-3′,5′-adenosinemonophosphate (cAMP) protein kinase. A (PKA) signaling pathway. Thesuperfamily of PDE isozymes consists of at least nine gene families(types): PDE1 to PDE9. Some PDE families are very diverse and consist ofseveral subtypes and numerous PDE isoform-splice variants. PDE isozymesdiffer in molecular structure, catalytic properties, intracellularregulation and location, and sensitivity to selective inhibitors, aswell as differential expression in various cell types.

A phosphodiesterase (PDE) inhibitor is defined herein as any drug usedin the treatment of pulmonary arterial hypertension that works byblocking the inactivation of cyclic AMP. There are five major subtypesof phosphodiesterase (PDE); the drugs enoximone (inhibits PDE IV) andmilrinone (Primacor®) (inhibits PDE IIIc) are most commonly usedmedically. Other phosphodiesterase inhibitors include Amrinone (Inocor®)used to improve myocardial function, pulmonary and systemicvasodilation, and sildenafil (Viagra®), tadalafil (Cialis®) andvardenafil (LEVITRA®)—selective phosphodiesterase V inhibitors.

Calcium Channel Blockers

In accordance with one embodiment of the present invention, a Rho-kinaseinhibitor, preferably fasudil, is administered in combination with asecond agent, which is a calcium channel blockers. Calcium channelblockers, or antagonists, act by blocking the entry of calcium intomuscle cells of heart and arteries so that the contraction of the heartdecreases and the arteries dilate. With the dilation of the arteries,arterial pressure is reduced so that it is easier for the heart to pumpblood. This also reduces the heart's oxygen requirement. Calcium channelblockers are useful for treating PPH. Due to blood pressure loweringeffects, calcium channel blockers are also useful to treat high bloodpressure. Because they slow the heart rate, calcium channel blockers maybe used to treat rapid heart rhythms such as atrial fibrillation.Calcium channel blockers are also administered to patients after a heartattack and may be helpful in treatment of arteriosclerosis.

Calcium channel blockers which are within the scope of this inventioninclude, but are not limited to: amlodipine (U.S. Pat. No. 4,572,909);bepridil (U.S. Pat. No. 3,962,238); clentiazem (U.S. Pat. No.4,567,175); diltiazem (U.S. Pat. No. 3,562,257); fendiline (U.S. Pat.No. 3,262,977); gallopamil (U.S. Pat. No. 3,261,859); mibefradil (U.S.Pat. No. 4,808,605); prenylamine (U.S. Pat. No. 3,152,173); semotiadil(U.S. Pat. No. 4,786,635); terodiline (U.S. Pat. No. 3,371,014);verapamil (U.S. Pat. No. 3,261,859); aranidipine (U.S. Pat. No.4,446,325); bamidipine (U.S. Pat. No. 4,220,649): benidipine (EuropeanPatent Application Publication No. 106,275); cilnidipine (U.S. Pat. No.4,672,068); efonidipine (U.S. Pat. No. 4,885,284); elgodipine (U.S. Pat.No. 4,952,592); felodipine (U.S. Pat. No. 4,264,611); isradipine (U.S.Pat. No. 4,466,972); lacidipine (U.S. Pat. No. 4,801,599); lercanidipine(U.S. Pat. No. 4,705,797); manidipine (U.S. Pat. No. 4,892,875);nicardipine (U.S. Pat. No. 3,985,758); nifedipine (U.S. Pat. No.3,485,847); nilvadipine (U.S. Pat. No. 4,338,322); nimodipine (U.S. Pat.No. 3,799,934); nisoldipine (U.S. Pat. No. 4,154,839); nitrendipine(U.S. Pat. No. 3,799,934); cinnarizine (U.S. Pat. No. 2,882,271);flunarizine (U.S. Pat. No. 3,773,939); lidoflazine (U.S. Pat. No.3,267,104); lomerizine (U.S. Pat. No. 4,663,325); bencyclane (HungarianPatent No. 151,865); etafenone (German Patent No. 1,265,758); andperhexyline (British Patent No. 1,025,578). The disclosures of all suchpatents and patent applications are incorporated herein by reference.

Preferred calcium channel blockers comprise amlodipine, diltiazem,isradipine, nicardipine, nifedipine, nimodipine, nisoldipine,nitrendipine, and verapamil, or, e.g., dependent on the specific calciumchannel blockers, a pharmaceutically acceptable salt thereof.

The compounds to be combined can be present as pharmaceuticallyacceptable salts. If these compounds have, for example, at least onebasic center, they can form acid addition salts. Corresponding acidaddition salts can also be formed having, if desired, an additionallypresent basic center. The compounds having at least one acid group (forexample COOH) can also form salts with bases. Corresponding internalsalts may furthermore be formed, if a compound of formula comprisese.g., both a carboxy and an amino group.

In accordance with one preferred embodiment of the present combinationtherapy, fasudil and iloprost are administered together with a secondgeneration calcium antagonist, such as amlodipine. The combination maybe administered in a sustained release dosage form. Preferably, thecombination dosage and release form is optimized for the treatment ofhypertensive patients.

Gleevec

Recently, Gleevec (Imatinib) has been shown to be successfully used intwo well established experimental models of progressive pulmonaryarterial hypertension. It was found that the treatment resulted invirtually complete reversal of lung vascular remodeling, pulmonaryhypertension and right heart hypertrophy.

In the Sep. 29, 2005 issue of the New England Journal of Medicine, a61-year-old man suffering from an advanced case of the disease saw hiscondition improve and stabilize after taking Gleevec (imatinib)—eventhough all other medications had failed. “Only the addition of Gleevecwas able to prevent further deterioration, and even improved hiscondition,” said co-researcher Dr. Hossein A. Ghofrani, of UniversityHospital Giessen, in Germany. Although a single case report does notwarrant widespread use of Gleevec for pulmonary hypertension, the Germanresearchers who wrote the report said they are now planning a largeclinical trial. Accordingly, Applicant considers Gleevec to be apotential additional ingredient in the combination therapies disclosedherein.

The therapeutic combinations disclosed herein for the treatment and/orprevention of PAH and/or stable angina are combinations of known drugs.The pharmacologic properties of these drugs are well known to theskilled practitioner. The combinations may be formulated or otherwiseindividually administered such that the relative amounts of each drug inthe combination is sufficient, when combined to treat and/or prevent atleast one symptom associated with PAH and/or stable angina. The symptomsof PAH and stable angina are well known. Moreover, animal models ofthese conditions may be used to optimize dosages (see e.g., thereferences cited and incorporated herein). The skilled practitioner willbe able to determine without undue experimentation optimal dosages. Itis likely that lower doses, for example of fasudil in combination withiloprost or sarpogrelate, may be used in the recited combinationsbecause the individual agents may interact in additive or synergisticmanners, and preferably, the individual agents target independentmechanisms of action.

It is understood that all drugs disclosed as potential candidates forthe recited combination therapies may be used in their free forms or assalts thereof. It is also understood that the recitation of therapeuticcombinations and methods of treatment include active metabolites, formedin vivo, which are known in the art for the disclosed candidate drugs.

The drugs in the recited therapeutic combinations can be administered,as described herein, according to any of a number of standard methodsincluding, but not limited to injection, suppository, nasal spray,time-release implant, transdermal patch, and the like. In particularlypreferred embodiments, at least one of the drugs in the combination isadministered orally (e.g. as a syrup, capsule, or tablet) or byinhalation (either as a dry powder formulation or as a nebulizedformulation). In one preferred embodiment, fasudil, hydroxyfasudil, orsalts thereof, may be given orally. More preferably, the fasudil,hydroxyfasudil, or salts thereof, is administered as a sustained-releaseformulation sufficient to provide extended plasma half-life and minimizethe peaks and troughs of plasma concentrations between dosing. Suchformulations are well known in the art and may enhance bioavailabilityof the drug. In another preferred embodiment, fasudil, hydroxyfasudil,or salts thereof, may be administered by inhalation.

More generally, injectable preparations include sterile suspensions,solutions or emulsions of the active ingredients in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing and/or dispersing agent. The formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmultidose containers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, stored preparations can be supplied in unit dosage formsand reconstituted prior to use in vivo.

For prolonged delivery, the active ingredients can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the candidate drugs.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch which slowly releases the active ingredient forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activeingredients.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. For rectal and vaginalroutes of administration, the active ingredients may be formulated assolutions (for retention enemas) suppositories or ointments.

For administration by inhalation, the active ingredients can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredients. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Example 1 Isolated PA Rings

Small (4th or 5th branch) pulmonary arteries (SPA, 200-300 μm ID) wereisolated from rats exposed to chronic hypoxia (˜10% oxygen) for 3 to 4weeks. Briefly, after anesthesia with pentobarbital sodium (30 mg ip)and heparinization (100 IU), the heart and lungs were removed en bloc,and the SPA were isolated under a dissecting microscope. Care was takento avoid damage to the endothelium. The SPA rings were, placed on steelwires attached to a force transducer and suspended in baths containing10 ml of physiological salt solution (PSS) at 37° C. Resting passiveforce was adjusted to a previously determined optimal tension(determined by maximum response to 80 mM KCl: 400 mg for SPA fromnormotensive lungs (NL), and 750 mg for SPA from hypoxic hypertensivelungs (HL). Rings were gassed with 21% O₂-5% CO₂-74% N₂ and allowed toequilibrate for 60 mM. The artery rings were then constricted with acombination of endothelin-1 (ET-1) (3 nM) plus thromboxane analogueU46619 (30 nM) plus 5-HT (3 μM). After the vasoconstrictor-inducedconstriction of the pulmonary artery rings had reached a plateau, thevarious vasodilators (iloprost, bosentan, sildenafil (Viagra™) orfasudil) were added individually to the muscle baths to achieve acalculated concentration. When the vasodilator response to the firstconcentration of drug had reached a plateau, additional vasodilator wasadded to the bath to achieve the next higher concentration, and so onuntil the highest concentration used was reached to obtain dose-responsecurves for each compound individually. Then, after the highestconcentrations of iloprost, bosentan, and sildenafil were tested,increasing concentrations of fasudil were added sequentially to the samebaths to obtain dose-response curves for fasudil in the presence of thetested compounds. FIGS. 1-3 show the obtained dose-response curves.

Example 2 Isolated PA Rings

The experiments are performed essentially as described above, but therings are constricted with each of the vasoconstrictors individually.The dose response curves of fasudil with iloprost, fasudil withbosentan, fasudil with sildenafil show synergistic effects on therelaxation of the PA rings.

Example 3 Conscious Catheterized Rats

Experiments are performed with a chronically hypoxic,pulmonary-hypertensive group of rats which have been exposed tohypobaric hypoxia (410 mmHg barometric pressure, 76 mmHg inspired O₂tension) for 3-4 wk in a chamber flushed continuously with room air toprevent accumulation of CO₂, NH₃, and H₂O. Hypobaric exposure has been24 h/day, except when the chamber has been opened for 10-15 min every 2days to remove rats or clean cages and replenish food and water. Allrats are exposed to a 12:12-h light-dark cycle and allowed free accessto standard rat food and water.

The chronically hypoxic rats are anesthetized with ketamine (100 mg/kg)and xylazine (15 mg/kg) for placement of catheters in the right jugularvein and pulmonary and right carotid arteries (Oka M. 2001 Am J PhysiolLung Cell Mol Physiol 280: L432-L435). The rats are allowed to recoverfor 48 h in room air. After recovery, conscious rats are placed in aventilated plastic box, and pulmonary and systemic arterial pressuresare measured with pressure transducers. Cardiac output is determined bya standard dye-dilution method, and total pulmonary resistance (TPR) iscalculated by dividing mean PA pressure (MPAP) by cardiac output. Therats are then injected with various doses of vasodilators, such asiloprost, bosentan or sildenafil alone or in combination with fasudil at30-min intervals between doses. Pressures are measured 10 min afterinjection of each dose. Cardiac output measurements are repeated 10 minafter injection of the lowest and the highest doses tested for eachcompound. After the final dose, rats are again exposed to 10 min ofhypoxia. The combinations show synergistic effects on the MPAP and TPRof the chronically hypoxic rats.

Example 4 Isolated Perfused Lungs

After pulmonary-hypertensive rats are removed from the hypobaric chamberand anesthetized with pentobarbital sodium (30 mg i.p.), lungs areisolated and perfused for vasoreactivity studies. The techniques of lungisolation, ventilation, and perfusion have been described in detailelsewhere (Morio Y. & McMurtry I. F. 2002 J Appl Physiol 92:527-534).The perfusate is 20 ml of PSS. Ficoll (4 g/100 ml; type 70, Sigma) isincluded as a colloid, and 3.1 μM meclofenamate (Sigma) is added toinhibit synthesis of vasodilator prostaglandins. Perfusion rate is 0.04ml-g body wt⁻¹ min⁻¹. PSS-perfused hypoxic hypertensive lungs (HL) areequilibrated at 37° C. for 20 min during ventilation with 8% O₂-5%CO₂-87% N₂. After equilibration, two hypoxic pressor responses areelicited by 10 min of ventilation with 0% O₂-5% CO₂-95% N₂ and 3% O₂-5%CO₂-92% N₂, separated by 10 min of normoxic ventilation, to inducehypoxic vasoreactivity. The drugs are added to the perfusate reservoirto achieve the calculated circulating concentrations. Thevasoconstriction is achieved by Nomega-nitro-L-arginine (L-NNA), a NOsynthase (NOS) inhibitor. After development of the L-NNA-inducedvasoconstriction, iloprost, bosentan, sildenafil, or fasudil alone, oriloprost, bosentan, sildenafil each in combination with fasudil areadded cumulatively to the perfusate of separate lungs at 10-minintervals. The baseline perfusion pressure is measured before and afterthe vasodilators. The combinations of the tested drugs showedsynergistic effects on the reduction of the baseline perfusion pressure.

While a number of preferred embodiments of the invention and variationsthereof have been described in detail, other modifications and methodsof using the disclosed therapeutic combinations will be apparent tothose of skill in the art. Accordingly, it should be understood thatvarious applications, modifications, and substitutions may be made ofequivalents without departing from the spirit of the invention or thescope of the claims. Further, it should be understood that the inventionis not limited to the embodiments set forth herein for purposes ofexemplification, but is to be defined only by a fair reading of theappended claims, including the full range of equivalency to which eachelement thereof is entitled.

REFERENCES

-   1. Kato et al. (2000) Respirology 5:-   2. Kato et al. (2000) J. Int. Med. Res. 28: 258-268.-   3. Doggrell (2004) Expert Opin. Investig. Drugs 13: 7865-874.-   4. Saini et al (2004) Cardiovasc. Drug Rev. 22; 27-54.-   5. Abe et al. (2005) J Cardiovasc Pharmacol 45:120-124.-   6. Nagaoka et al. (2004) Am. J. Physiol. Lung Cell Mol. Physiol.    287: L665-L672.-   7. Fukumoto et al. (2005) Heart 91: 391-392.-   8. Takemoto et al. (2002) Circulation 106: 57-62.-   9. Abe et al. (2004) Circ Res 94: 385-393.-   10. Tramontano et al. (2004) Biochem Biophys Res Comm 320: 34-38.-   11. Ohnaka et al. (2001) Biochem Biophys Res Comm 287: 337-342.-   12. Shimokawa et al. (1999) Cardiovasc. Res. 43:1029-1039,-   13. Hirooka et al. (2005) Am J Cardiovasc Drugs 5(1): 31-39.

All of the references cited herein are incorporated in their entirety byreference thereto.

1. A therapeutic combination, comprising an effective amount of fasudiland sildenafil.
 2. The therapeutic combination of claim 1, wherein thefasudil and sildenafil are formulated independently.
 3. The therapeuticcombination of claim 1, wherein the fasudil is formulated foradministration via inhalation.
 4. The therapeutic combination of claim3, wherein the fasudil is formulated in a dry powder or an aerosolizablesolution.
 5. A method for treating pulmonary arterial hypertension in aperson in need of such treatment comprising administering a therapeuticcombination of fasudil and sildenafil according to claim
 1. 6. Themethod according to claim 5, wherein the fasudil and sildenafil areadministered in separate dosage forms.
 7. The method according to claim5, wherein the fasudil is administered by inhalation.
 8. The methodaccording to claim 7, wherein the fasudil is administered in a drypowder or an aerosolizable solution.